Biosynthesis of silver nanoparticles by a new strain of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. compared with <Emphasis Type="Italic">Aspergillus</Emphasis><Emphasis Type="Italic">fumigatus</Emphasis>

Locally isolated strains of a thermoalkalotolerant Streptomyces sp. and Aspergillus fumigatus were used for the in vitro biosynthesis of silver nanoparticles from AgNO₃ solutions. An autolysed cell-free culture filtrate from each strain was used, indicating that the formation mechanism depends on intra-cellular components for both organisms, since culture broths had no significant nanoparticle formation potential. Nanoparticle formation was indicated by a change of the solution from colourless or light brown to dark brown after 24 h or more, and UV–visible spectroscopy and x-ray diffraction analysis confirmed the formation by both organisms. The initial formation kinetics were faster with Aspergillus, but formation continued for a longer period with Streptomyces, resulting in higher concentrations after 48 h. Transmission electron microscope images revealed well dispersed nanoparticles with diameters ranging from 15 to 45 nm from A. fumigatus, while those from Streptomyces sp. had a narrower size distribution of 15–25 nm. The higher productivity and preferred narrower size distribution of Streptomyces, together with its well established industrial use, may make it the preferred choice for further optimization studies.     

<Emphasis Type="Italic">Streptomyces</Emphasis><Emphasis Type="Italic">mayteni</Emphasis> sp. nov., a novel actinomycete isolated from a Chinese medicinal plant

An actinomycete strain, designated YIM 60475^T, was isolated from the roots of Maytenus austroyunnanensis and was characterized by using a polyphasic approach. The strain was determined to belong to the genus Streptomyces, based on its phenotypic and phylogenetic characteristics. The strain produced spiral spore chains on aerial mycelium. The cell wall contained ll-diaminopimelic acid. Whole-cell hydrolysates contained galactose, glucose, and xylose. The phospholipid was type II. The DNA G+C content of the type strain was 73.3 mol%. DNA–DNA hybridization and comparison of physiological and chemical characteristics suggested that strain YIM 60475^T is a new Streptomyces species, for which the name Streptomyces mayteni sp. nov. is proposed. The type strain is YIM 60475^T (=CCTCC AA 207005^T = KCTC 19383^T). Hua-Hong Chen and Sheng Qin contributed equally to this work.     

Isolation and characterization of an efficient nitro-reducing bacterium, <Emphasis Type="Italic">Streptomyces</Emphasis><Emphasis Type="Italic">mirabils</Emphasis> DUT001, from soil

A bacterial strain, designated as DUT001, was isolated from soil samples collected in Qingpu, Shanghai, China, and found to be capable of reducing nitro polycyclic aromatic compounds and polynitrated aromatic compounds. It was identified as Streptomyces mirabils based on morphological and physiological-biochemical properties, as well as 16S rDNA sequences and phylogenetic analysis. The optimum growth temperature and pH for Streptomyces mirabils DUT001 are 28°C and 7.2, respectively. The best carbon source and nitrogen source are glucose and yeast extract. Streptomyces mirabils DUT001 exhibited highly efficient activity in reduction of nitroaromatic compounds, 4-nitro-1, 8-naphthalic anhydride (4-NNA), 3-nitro-1, 8-naphthalic anhydride, 4-nitrophthalimide, 3-nitrophthalimide 1, 3-dinitrobenzene, and 1, 4-dinitrobenzene, to their amino derivatives with yields ranging from 70–99% under mild conditions. 1% (w/v) of the nonionic surfactant, Triton X-100 and 0.1 mM of redox mediator, anthraquinone-2, 6-disulfonate (AQDS), could accelerate the nitroreduction process. The bioreduction activity of the Streptomyces mirabils DUT001 offers its application potentials in bioremediation of nitroaromatic compounds and synthesis of important amino derivatives.     

Extracellular production of a sphingomyelinase from <Emphasis Type="Italic">Streptomyces griseocarneus</Emphasis> using <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

The structural gene for sphingomyelinase (SMase) from Streptomyces griseocarneus, was introduced into Streptomyces lividans using a shuttle vector, pUC702, for Escherichia coli/S. lividans. High-level secretory production of SMase was achieved using the promoter, signal sequence and terminator regions of phospholipase D from Streptoverticillium cinnamoneum. The transformant constitutively expressed a high specific activity of SMase extracellularly during batch culture. Maximum SMase activity (555 ± 114 U/mg protein) was with 1.75 M MgCl₂ which was about 50-fold more than that with 10 mM MgCl₂.     

Proteomic approach to enhance doxorubicin production in <Emphasis Type="Italic">panK</Emphasis>-integrated <Emphasis Type="Italic">Streptomyces peucetius</Emphasis> ATCC 27952

Biosynthesis of polyketide compounds depends upon the starter and extender units of coenzyme A derivatives of carboxylic acids present in the host organism. To increase the coenzyme A (CoA) pool, pantothenate kinase (panK) gene from Escherichia coli was integrated into S. peucetius ATCC 27952 (panK-integrated strain, BG200), which resulted in increase in aglycone polyketide ε-rhodomycinone (RHO), but decrease in the desired product, i.e., doxorubicin (DXR). To reduce RHO accumulation by synthesizing daunorubicin (DNR) from RHO more efficiently, glycosyltransferase (dnrQS) was overexpressed (pIBR25::dnrQS in panK-integrated strain, BG201). However, DnrQS overexpression still resulted in less production of DXR compared with the parental strain. To understand the results in detail by investigating the proteome changes in the panK-integrated strain, two-dimensional (2D) gel electrophoresis was performed. Among the several proteins that are up- or downregulated in BG200, efflux protein DrrA was our main target of interest, because it is directly related to DXR/DNR production in S. peucetius. DXR transporter DrrAB was additionally introduced in BG200 to enhance secretion of toxic DXR. Compared with S. peucetius ATCC 27952, BG204 (pIBR25::drrAB in panK-integrated strain), produced two times higher amount of DXR, which is 9.4-fold higher than that of panK-integrated strain BG200. The results show that the proteomic approach is quite useful in host development of Streptomyces and understanding cell physiology for antibiotic production.     

Two heterologously expressed <Emphasis Type="Italic">Planobispora rosea</Emphasis> proteins cooperatively induce <Emphasis Type="Italic">Streptomyces lividans</Emphasis> thiostrepton uptake and storage from the extracellular medium

Background  

A bacterial artificial chromosomal library of Planobispora rosea, a genetically intractable actinomycete strain, was constructed using Escherichia coli - Streptomyces artificial chromosome (ESAC) and screened for the presence of genes known to be involved in the biosynthesis of antibiotics.     

Initial impacts of <Emphasis Type="Italic">Microcystis</Emphasis><Emphasis Type="Italic">aeruginosa</Emphasis> blooms on the aquatic food web in the San Francisco Estuary

The impact of the toxic cyanobacterium Microcystis aeruginosa on estuarine food web production in San Francisco Estuary is unknown. It is hypothesized that Microcystis contributed to a recent decline in pelagic organisms directly through its toxicity or indirectly through its impact on the food web after 1999. In order to evaluate this hypothesis, phytoplankton, cyanobacteria, zooplankton, and fish were collected biweekly at stations throughout the estuary in 2005. Concentrations of the tumor-promoting Microcystis toxin, microcystin, were measured in water, plankton, zooplankton, and fish by a protein phosphatase inhibition assay, and fish health was assessed by histopathology. Microcystis abundance was elevated in the surface layer of the western and central delta and reached a maximum of 32 × 10⁹ cells l⁻¹ at Old River in August. Its distribution across the estuary was correlated with a suite of phytoplankton and cyanobacteria species in the surface layer and 1 m depth including Aphanizomenon spp., Aulacoseira granulata, Bacillaria paradoxa, Rhodomonas spp., and Cryptomonas spp. Shifts in the phytoplankton community composition coincided with a decrease in the percentage of diatom and green algal carbon and increase in the percentage of cryptophyte carbon at 1 m depth. Maximum calanoid and cyclopoid copepod carbon coincided with elevated Microcystis abundance, but it was accompanied by a low cladocera to calanoid copepod ratio. Total microcystins were present at all levels of the food web and the greater total microcystins concentration in striped bass than their prey suggested toxins accumulated at higher trophic levels. Histopathology of fish liver tissue suggested the health of two common fish in the estuary, striped bass (Morone saxatilis), and Mississippi silversides (Menidia audens), was impacted by tumor-promoting substances, particularly at stations where total microcystins concentration was elevated. This study suggests that even at low abundance, Microcystis may impact estuarine fishery production through toxic and food web impacts at multiple trophic levels.     

Novel metabolic pathway for salicylate biodegradation via phenol in yeast <Emphasis Type="Italic">Trichosporon moniliiforme</Emphasis>

A novel metabolic pathway was found in the yeast Trichosporon moniliiforme WU-0401 for salicylate degradation via phenol as the key intermediate. When 20 mM salicylate was used as the sole carbon source for the growth of strain WU-0401, phenol was detected as a distinct metabolite in the culture broth. Analysis of the products derived from salicylate or phenol through reactions with resting cells and a cell-free extract of strain WU-0401 indicated that salicylate is initially decarboxylated to phenol and then oxidized to catechol, followed by aromatic ring cleavage to form cis-cis muconate.     

<Emphasis Type="Italic">Streptomyces fildesensis</Emphasis> sp. nov., a novel streptomycete isolated from Antarctic soil

A novel actinomycete strain, GW25-5^T, was isolated from a soil sample collected from the Fildes Peninsula, King George Island, West Antarctica. The strain was characterized by white to grey aerial mycelia, which were differentiated to straight to flexuous spore chains, with rod-shaped smooth spores. The cell wall of strain GW25-5^T contained LL-diaminopimelic acid (A₂pm) and traces of meso-A₂pm. Whole-cell sugars were galactose and minor amounts of mannose and glucose. The predominant menaquinones were MK-9(H₆) (49%), MK-9(H₈) (24%) and MK-9(H₄) (12%). The phospholipids contained DPG, PE, PI, PIM and PL(s). The major cellular fatty acids were iso-C_16:0 and anteiso-C_15:0. Genomic DNA G+C content of strain GW25-5^T was 70.0 mol%. BLAST result showed that strain GW25-5 has the 16S rRNA gene sequence highest similarity of 97.5% with members of genus Streptomyces and phylogenetic analysis indicated that this strain belongs to the genus Streptomyces. DNA–DNA relatedness values of strain GW25-5^T with the closest species of Streptomyces purpureus LMG 19368^T and Streptomyces beijiangensis YIM 6^T were significantly lower than 70% of the threshold value for the delineation of genomic species. A polyphasic taxonomic investigation based on a judicious combination of genotypic and phenotypic characteristics revealed that the organism represents a novel species of the genus Streptomyces. Thus, we propose strain GW25-5^T as the type strain of this novel species, Streptomyces fildesensis (=CGMCC 4.5735^T = YIM 93602^T = DSM 41987^T = NRRL B 24828^T).     

Cyanobactericidal effect of <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. isolated from eutrophic lake on <Emphasis Type="Italic">Microcystis</Emphasis> sp

A bacterium, which was observed in all cultivations of Microcystis sp., was isolated and designated as Rhodococcus sp. KWR2. The growth of bloom-forming cyanobacteria, including four strains of Microcystis aeruginosa and Anabaena variabilis, was suppressed by up to 75–88% by 2% (v/v) culture broth of KWR2 after 5 days. But KWR2 did not inhibit eukaryotic algae, Chlorella vulgaris and Scenedesmus sp. An extracellular algicidal substance produced by KWR2 showed a cyanobactericidal activity of 94% and was water-soluble with a molecular weight of lower than 8 kDa.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Prevalence of genetic differences in phosphorylcholine expression between nontypeable <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> and <Emphasis Type="Italic">Haemophilus haemolyticus</Emphasis>

Background  

Although non-typeable (NT) Haemophilus influenzae and Haemophilus haemolyticus are closely related human commensals, H. haemolyticus is non-pathogenic while NT H. influenzae is an important cause of respiratory tract infections. Phase-variable phosphorylcholine (ChoP) modification of lipooligosaccharide (LOS) is a NT H. influenzae virulence factor that, paradoxically, may also promote complement activation by binding C-reactive protein (CRP). CRP is known to bind more to ChoP positioned distally than proximally in LOS, and the position of ChoP within LOS is dictated by specific licD alleles (designated here as licD _I , licD _III , and licD _IV ) that are present in a lic1 locus. The lic1 locus contains the licA-licD genes, and ChoP-host interactions may also be influenced by a second lic1 locus that allows for dual ChoP substitutions in the same strain, or by the number of licA gene tetranucleotide repeats (5'-CAAT-3') that reflect phase-variation mutation rates.     

Overproduction of soluble recombinant transglutaminase from <Emphasis Type="Italic">Streptomyces netropsis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A novel microbial transglutaminase (TGase) from the cultural filtrate of Streptomyces netropsis BCRC 12429 (Sn) was purified. The specific activity of the purified TGase was 18.2 U/mg protein with an estimated molecular mass of 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The TGase gene of S. netropsis was cloned and an open reading frame of 1,242 bp encoding a protein of 413 amino acids was identified. The Sn TGase was synthesized as a precursor protein with a preproregion of 82 amino acid residues. The deduced amino acid sequence of the mature S. netropsis TGase shares 78.9–89.6% identities with TGases from Streptomyces spp. A high level of soluble Sn TGase with its N-terminal propeptide fused with thioredoxin was expressed in E. coli. A simple and efficient process was applied to convert the purified recombinant protein into an active enzyme and showed activity equivalent to the authentic mature TGase. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Increase in Insecticidal Toxicity by Fusion of the <Emphasis Type="Italic">cry1Ac</Emphasis> Gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> with the Neurotoxin Gene <Emphasis Type="Italic">hwtx-I</Emphasis>

A fusion gene was constructed by combining the cry1Ac gene of Bacillus thuringiensis strain 4.0718 with a neurotoxin gene, hwtx-1, which was synthesized chemically. In this process, an enterokinase recognition site sequence was inserted in frame between two genes, and the fusion gene, including the promoter and the terminator of the cry1Ac gene, was cloned into the shuttle vector pHT304 to obtain a new expression vector, pXL43. A 138-kDa fusion protein was mass-expressed in the recombinant strain XL002, which was generated by transforming pXL43 into B. thuringiensis acrystalliferous strain XBU001. Quantitative analysis indicated that the expressed protein accounted for 61.38% of total cellular proteins. Under atomic force microscopy, there were some bipyramidal crystals with a size of 1.0 × 2.0 μm. Bioassay showed that the fusion crystals from recombinant strain XL002 had a higher toxicity than the original Cry1Ac crystal protein against third-instar larvae of Plutella xylostella, with an LC₅₀ (after 48 h) value of 5.12 μg/mL. The study will enhance the toxicity of B. thuringiensis Cry toxins and set the groundwork for constructing fusion genes of the B. thuringiensis cry gene and other foreign toxin genes and recombinant strains with high toxicity. LiQiu Xia and XiaoShan Long contributed equally to this work.     

Nematicidal activity of fervenulin isolated from a nematicidal actinomycete, <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CMU-MH021, on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>

An isolate of the actinomycete, Streptomyces sp. CMU-MH021 produced secondary metabolites that inhibited egg hatch and increased juvenile mortality of the root-knot nematode Meloidogyne incognita in vitro. 16S rDNA gene sequencing showed that the isolate sequence was 99% identical to Streptomyces roseoverticillatus. The culture filtrates form different culture media were tested for nematocidal activity. The maximal activity against M. incognita was obtained by using modified basal (MB) medium. The nematicidal assay-directed fractionation of the culture broth delivered fervenulin (1) and isocoumarin (2). Fervenulin, a low molecular weight compound, shows a broad range of biological activities. However, nematicidal activity of fervenulin was not previously reported. The nematicidal activity of fervenulin (1) was assessed using the broth microdilution technique. The lowest minimum inhibitory concentrations (MICs) of the compound against egg hatch of M. incognita was 30 μg/ml and juvenile mortality of M. incognita increasing was observed at 120 μg/ml. Moreover, at the concentration of 250 μg/ml fervenulin (1) showed killing effect on second-stage nematode juveniles of M. incognita up to 100% after incubation for 96 h. Isocoumarin (2), another bioactive compound produced by Streptomyces sp. CMU-MH021, showed weak nematicidal activity with M. incognita.     

<Emphasis Type="Italic">Streptomyces lividans</Emphasis> and <Emphasis Type="Italic">Brevibacterium lactofermentum</Emphasis> as heterologous hosts for the production of X<Subscript>22</Subscript> xylanase from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>

The Aspergillus nidulans gene xlnA coding for the fungal xylanase X₂₂ has been cloned and expressed in two heterologous bacterial hosts: Streptomyces lividans and Brevibacterium lactofermentum. Streptomyces strains yielded 10 units/ml of xylanase when the protein was produced with its own signal peptide, and 19 units/ml when its signal peptide was replaced by the one for xylanase Xys1 from Streptomyces halstedii. B. lactofermentum was also able to produce xylanase X₂₂, affording 6 units/ml upon using either the Aspergillus xlnA signal peptide or Streptomyces xysA. These production values are higher than those previously reported for the heterologous expression of the A. nidulans xlnA gene in Saccharomyces cerevisiae (1 unit/ml). Moreover, the X₂₂ enzyme produced by Streptomyces lividans showed oenological properties, indicating that this Streptomyces recombinant strain is a good candidate for the production of this enzyme at the industrial scale.     

Rhizospheric streptomycetes as potential biocontrol agents of <Emphasis Type="Italic">Fusarium</Emphasis> and <Emphasis Type="Italic">Armillaria</Emphasis> pine rot and as PGPR for <Emphasis Type="Italic">Pinus taeda</Emphasis>

Pinus taeda is one of the main timber trees in Brazil, occupying 1.8 million ha with an annual productivity of 25–30 m³ ha⁻¹. Another important species is Araucaria angustifolia, belonging to the fragile Rainforest biome, which for decades has been a major source of timber in Brazil. Some diseases that affect the roots and/or the stem of these trees and cause “damping-off” of the seedlings, with economic and environmental losses for the forest sector, are caused by the plant pathogenic fungi Fusarium sp. or Armillaria sp. This research project intended to isolate actinobacteria from the Araucaria rhizosphere, which present an antagonistic effect against these fungi. After the selection of the best pathogen inhibitors, morphologic characteristics, enzyme production, and their effect on the growth of Pinus taeda were studied. The actinobacteria were tested for their antagonistic capacity against Fusarium sp. in Petri plates with PDA as substrate. The inhibition zone was measured after 3, 5, 7, and 10 days. Of all the isolates tested, only two of them maintained inhibition zones up to 4 mm for 10 days. The inhibition of Armillaria sp. was tested in liquid medium and also in Petri dishes through the evaluation of the number of the fungal rhizomorphs in dual culture with the actinobacteria. It was found that all five isolates were able to inhibit the rhizomorph production, with the best performance of the isolate A43, which was capable of inhibiting both fungi, Fusarium and Armillaria. In a greenhouse experiment, the effect of five isolates on the growth of Pinus taeda seedlings was tested. Plant height, stem diameter, root and shoot dry matter were determined. The Streptomyces isolate A43 doubled plant growth. These results may lead to the development of new technologies in the identification of still unknown bacterial metabolites and new management techniques to control forest plant diseases.     

<Emphasis Type="Italic">Amycolatopsis endophytica</Emphasis> sp. nov., a novel endophytic actinomycete isolated from oil-seed plant <Emphasis Type="Italic">Jatropha curcas</Emphasis> L.

A novel actinomycete, designated KLBMP 1221^T, was isolated from the surface-sterilized seeds of an oil-seed plant Jatropha curcas L. collected from Sichuan Province, south-west China and was characterized taxonomically by using a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequence showed that this strain formed a distinct phyletic line within the radiation of the genus Amycolatopsis. The 16S rRNA gene sequence similarity indicated that strain KLBMP 1221^T was most closely related to Amycolatopsis eurytherma NT202^T (98.9%), Amycolatopsis tucumanensis ABO^T (98.8%), Amycolatopsis thermoflava N1165^T (98.6%) and Amycolatopsis methanolica IMSNU 20055^T (98.5%). Strain KLBMP 1221^T had morphological and chemotaxonomic properties that were consistent with its classification in the genus Amycolatopsis. However, DNA–DNA relatedness data and phenotypic differences clearly distinguished the isolate from its closest relatives. Based on the combined genotypic and phenotypic evidence, it is proposed that strain KLBMP 1221^T be classified as representative of a novel species for which the name Amycolatopsis endophytica sp. nov. is proposed. The type strain is KLBMP 1221^T (= KCTC 19776^T = CCTCC AA 2010003^T).